Difference between revisions of "Part:BBa K2933214"
(→‎Usage and Biology)
 
(3 intermediate revisions by the same user not shown)
Line 17: Line 17:
 
<partinfo>BBa_K2933214 parameters</partinfo>
 
<partinfo>BBa_K2933214 parameters</partinfo>
 
<!-- -->
 
<!-- -->
−
==Usage and Biology===
+
==Usage and Biology==
−
This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein SHD. It encodes a protein which is SHD fused with His tag. The fusion protein is about 29.0 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SHD .  It is convenient for us to purify our target protein.<br>
+
This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein SHD. It encodes a protein which is SHD fused with His tag. Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag.The fusion protein is about 29.0 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SHD .  It is convenient for us to purify our target protein.<br>
 
===Molecular cloning===
 
===Molecular cloning===
−
First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
+
First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:SHD-PCR1.png|500px]]<br>
 
   [[File:SHD-PCR1.png|500px]]<br>
Line 26: Line 26:
 
</p>
 
</p>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
−
 
−
 
−
===Expression and purification===
 
−
'''Pre-expression:'''<br>
 
−
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
−
'''Massive expressing:'''<br>
 
−
After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.<br>
 
−
 
−
'''Affinity Chromatography:'''<br>
 
−
We used the GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein. <br>
 
−
* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
 
−
* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
 
−
* Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
 
−
* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
 
−
* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
 
−
<p style="text-align: center;">
 
−
    [[File:T--TJUSLS China--SHD GST.jpg|400px]]<br>
 
−
 
−
'''Figure 2.'''  The result of SDS-page.<br>
 
−
</p>
 

Latest revision as of 14:03, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+SHD+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+SHD），and the biological module can be built into E.coli for protein expression

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein SHD. It encodes a protein which is SHD fused with His tag. Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag.The fusion protein is about 29.0 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SHD . It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

SHD-PCR1.png
Figure 1. The PCR result of SHD.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.